This invention relates to a method for the excitation of long-lived fluorescent and phosphorescent dyes.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
The method according to this invention allows the excitation of dyes emitting long-lived fluorescence and phosphorescence, with light of a wavelength considerably longer than their normal excitation wavelength. In the method according to the invention the dyes are excited by so-called two-photon excitation, the excitation resulting from the summation of the energies of two photons. The method according to this invention is applicable to fluorescent or phosphorescent dyes with an emission half-time longer than 100 ns due to an intermediary energetic triplet state. These dyes consequently have considerably longer half-times of excitation states than the emission half-times of ordinary organic dyes. The emission half-time of organic dyes is usually 1-10 ns, maximally 100 ns because emission usually does not occur via an intermediary triplet state.
These fluorescent and phosphorescent dyes are used in biospecific assays as molecular labels. Examples of the most widely used biospecific assay techniques are the immunoassay and the DNA hybridization assay. Binding of antibody is assayed by measuring the emitted light. Biospecific assays are used in in vitro diagnostics as well as in microscopy. In microscopy antibodies allow the detection and localization of e.g. the structural elements of micro-organisms.
Two-photon excitation of short-lived fluorescent molecules is a well-known technique e.g. in the fields of spectroscopy and microscopy. The said method requires, with current technology, high-energy laser instruments producing ultra-short pulses. Two-photon excitation is possible with a high momentary photon density. Simultaneous absorption of two photons is then probable. A high photon density is achieved by high luminous energy and by optical focusing of the light. Two-photon excitation has been described theoretically as early as 1931 (Goppert-Mayer, M. Ann. Phys. 1931, 9:273). The first prooves of the functionality of the method were obtained in the 1960's when laser instruments became available. In 1963 the first two-photon excitation in organic crystals was reported (Peticolas W. L., Goldsborough J. P., Reickhoff K. E. Phys. Rev. Let. 1963, Vol. 10/2). Two-photon excitations may also be observed with continuous high-energy laser radiation (Sepaniak M. J., Yeung ES. Anal. Chem. 1977, 49:1554-1556), but in this case the scattering of excitation light and heating of the sample interfere severely with measurement.
The advantage of two-photon excitation lies in the fact that visible light can be used for excitation instead of UV excitation. When visible light is used in order to elicit emission by exciting the dye by two simultaneous photon pulses, the scattering of light is reduced considerably as compared to excitation by UV radiation. In addition, two-photon excitation reduces damage caused by light to the sample below and above the object under examination. Two-photon excitation is best suited for the examination of small sample volumes or structures.
When applied to scanning microscopy, two-photon excitation allows a three-dimensional resolution comparable to the confocal microscope, without the second pinhole required in the confocal microscope. The method has been described in U.S. Pat. No. 5,034,613; 1991. The delimitation of the excitation in the three-dimensional space has been described in the literature (Anal. Chem. 1990, 62:973-976; Science, 1990, Vol. 248:73-76).
It is well known that the absorption of a single photon in a dye is, according to the concepts of probability, an independent event. The absorption of photons is a series of single, independent events. The probability of the absorption of a photon may be represented by a linear function. Absorption is linear as long as the energy states to be excited are not filled. It is known that the absorption of two or more photons is a nonlinear process (U.S. Pat. No. 5,034,613). When two or more photons are absorbed the absorption of single photons is no longer independent. A dye is excited only upon simultaneous absorption of all photons. The probability of the absorption of several photons is equal to the product of the probabilities of absorption of single photons. The emission caused by two photons is thus an exponential function to the power of two, the emission caused by three photons an exponential function to the power of three, etc.
The properties of an optical system for microimaging may be described by considering the response of the system to a point-like light source. A point-like light source forms, due to diffraction, an intensity distribution, characteristic of an optical system, in the focusing point (point response). This intensity distribution further reflects the resolution of the system. When normalized, this intensity distribution constitutes a probability distribution of the photons emitted from the point-like light source and hitting the focusing area. The nonlinear nature of two-photon absorption can be exploited to improve resolution. The probability distribution of two-photon excitation is then the normalized product of the intensity distributions of the first and the second photon. A probability distribution obtained in this way is clearly more delimited in the 3-dimensional space, especially in depth, than the probability ditribution of a single photon. Consequently, with two-photon excitation only the fluorescence generated in the clearly delimited, three-dimensional immediate vicinity of the focal point is detected (U.S. Pat. No. 5,034,613). The system is thus in its principle similar to the more traditional 3-dimensional optical microscope, or confocal microscope. In the confocal microscope the point response of the system is the normalized product of the probability distributions of point-like excitation and point-like detection (Confocal Microscopy, T. Wilson (ed), Academic Press, London, 1990, 1-64).
In order for the two photons to absorb, the dye must absorb the photons in such a way that the total sum of the energies of the photons equals the energy required for excitation (FIG. 1). The excitation then proceeds either via the intermediary energy state (omega 1) of the electrons of the molecule or directly to the excitation state (omega 2). The different possibilities of double-photon excitation are displayed in FIGS. 1A and 1B: in Figure A, directly as the combined result of two photons (lambda 1 and lambda 2); in Figure B, via the intermediary energy state (omega 1). Excitation via an intermediary state (FIG. 1B) requires a dye having such an intermediary energy state. Direct excitation of two photons according to FIG. 1A requires no intermediary state, instead the photons must absorb simultaneously, within approximately 10.sup.-15 seconds, in the same chromophor of the dye.
A disadvantage of current systems based on two-photon excitation is the cost of the extremely high-power pulsed laser and the large size and complexity of the equipment. A continuous laser requires continuous power of several watts. This destroys nearly all known organic samples. A further disadvantage is the scant amount of emitted light compared to excitation light, and scattering of excitation light in addition interferes with the detection of the emission from ordinary short-lived dyes.
The excitation states of excited dyes discharge their energy in an exponential relation according to equation [1]: EQU N=N.sub.0 exp(-t/tau) [1]
N is the number of discharging molecules at a given time point, N.sub.0 is the number of molecules discharging at time point=0, and tau is the average life-time of the excitation states ("decay parameter"). In the following discussion the terms long-lived and short-lived dyes are used to designate dyes with a long (100 ns-10 ms) or a short (1 ns-100 ns) average life-time tau.
Time-resolved detection of long-lived dyes involves the use of pulsed light for the excitation of the dye, and detection is started after a delay with respect to the excitation pulse. Detection is typically by photon counting, the counting being done within a specified time window. When detecting lanthanide chelates the photons are counted within e.g. 0.1-1 ms after the termination of the excitation pulse.
It is well known that time-resolved fluorescence detection of long-lived fluorescent dyes allows a sensitivity greater by several orders of magnitude than is possible in the detection of short-lived fluorescent molecules. The said molecules have allowed e.g. partial replacement of radioisotopes in immunodiagnostics and improvement of the sensitivity of detection (Soini, Lovgren. CRC Crit. Rev. Anal. Chem. 1987, 18:105-154).
Also in microscopy time-resolved detection of long-lived fluorescence allows reduction of background and thus greater sensitivity (Seveus et al. Cytometry, 1990, 13:329-338).
Long-lived fluorescence required in time-resolved detection is a property of e.g. the ions Eu.sup.3+, Sm.sup.3+, and Tb.sup.3+ of the lanthanide group. In inorganic compounds, the absorption properties of these metals are quite inferior, but when an organic ligand is linked to the metal, absorption can be improved considerably. In these so-called lanthanide chelates the photon to be excited is absorbed in the organic ligand, from which the excitation energy is transferred to the lanthanide ion. The excitation of all known lanthanides occurs in UV area 250 nm-370 nm. The structure and use of lanthanide chelates has been described by e.g. Ilkka Hemmila in his book: Applications of Fluorescence in Immunoassays: 7.4.1 (140-145), John Wiley & Sons, N.Y., 1991.
A known disadvantage of UV-excited long-lived dyes like the lanthanide chelates is the scattering of light in the UV area, which interferes with the detection of emission. Also the manufacturing of UV-transparent components functioning correctly with UV light is difficult. Neither is the functioning and stability of the flashlamps currently used in immunoassays satisfactory in all respects. Consequently, it will be advantageous to design a system capable of employing excitation light of longer wavelength.
When ultrashort pulses (&lt;1 ps) described in U.S. Pat. No. 5,034,613 are used for two-photon excitation, the life-time of the dye is without significance because the life-time of the short-lived or long-lived light-emitting dyes is in any case longer by orders of magnitude than the life-time of the exciting pulse. During the ultrashort pulse the energy states will be excited independently of the life-time of the emission of the dye. Ultrashort pulses are advantageous when using short-lived fluorescent molecules, as they allow control of average energy and damage to the sample. The scattering of light detected by the measuring instruments is also the least with ultrashort pulses. The dependence of the fluorescence intensity (I) obtained with two-photon excitation, on the excitation mechanism may be modelled by the equation: EQU I=Constant*P.sup.2 t [2]
P is the maximum power of the pulsed excitation light, and t is the duration of the pulse. The constant is dependent on the optical system and the dye used. This principle has been described in the literature (Wirth, Fatumbi. Anal. Chem. 1990, 62:973-976). This principle can also be deduced by the methods of probability calculation.
In two-photon excitation of short-lived molecules the objective is to achieve as short pulses as possible, of maximal power, to keep the energy of the pulses according to equation [2] as low as possible.